Abstract

The ultimate goal of proteomics is to understand complex biological systems. The first step toward this end is the discovery of protein differences by profiling a given proteome. One approach to proteome profiling is to fractionate it into intact proteins, with subsequent identification and quantitation. In this work, lysates of bovine skeletal muscle were prepared. Reproducible proteome profiles were generated by an automatic two-dimensional protein fractionation system. Proteins were separated by isoelectric point and then by hydrophobicity. The data collected from both separations were used to generate proteome profiles. A high protein content fraction with pl above 8.5 was digested with trypsin, and its main protein component was identified as lysozyme C by matrix assisted laser desorption/ionization–time of flight mass spectrometry.

The proteome profile of bovine skeletal muscle is of great interest to the meat industry because the muscle composition can have an impact on the meat tenderness and flavor.1 Previous profiles of bovine skeletal muscle reported in the literature have been done with two-dimensional (2D) gel electrophoresis.1–3 This technique separates proteins by isoelectric focusing (IEF) and molecular weight. One of its disadvantages is that proteins with isoelectric point (pI) values above and below the IEF pH range are lost and not detected. Other disadvantages of 2D gels include (a) the time required for preparation and analysis and (b) the caution needed while extracting the proteins from the gel.

This study shows the first proteome profile of bovine skeletal muscle generated by a 2D protein fractionation system based on liquid chromatography. In this system, the proteins were separated and fractionated by their pI values (first dimension) and by hydrophobicity (second dimension) in less than 24 h. Proteins with pI values above and below the separation pH range were collected and analyzed in the second dimension. The data collected from both separations were used to generate a proteome profile of pI versus reversed-phase retention time. A second-dimension fraction with a pI value above 8.5 and high protein content was digested with trypsin and successfully identified by matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) as lysozyme C.

MATERIALS AND METHODS

Reagents

All chemicals were from Sigma (St. Louis, MO), unless specified otherwise. High-quality water from an E-pure deionizer (Barnstead Thermolyne, Dubuque, IA) was used in the preparation of all solutions and in all procedures that required water.

Preparation of Bovine Skeletal Muscle Lysate

Three 100-mg samples (A, B, and C) from the same bovine skeletal leg muscle (acquired from a supermarket) were stored at −80°C and lysed on different days using the following protocol: The muscle sample was permitted to reach room temperature and minced with a clean razor blade. Then it was rinsed three times with Dulbecco’s phosphate-buffered saline (Invitrogen, Carlsbad, CA), after which the excess liquid was removed by suction. Lysis buffer was added to the muscle sample up to a final volume of 2.5 mL. The composition of the lysis buffer was 5 M urea, 10% glycerol, 2 M thiourea, 2.5% (w/v) SB3-10, 50 mM Tris-HCl, 5 mM Tris (2-carboxyethyl) phosphine hydrochloride, 2% (w/v) n-octylglucoside, and 1 mM protease inhibitor cocktail. The sample was then frozen at −20°C and thawed at 37°C for three cycles, with vigorous vortexing after each thawing. Next it was centrifuged at 20,000 g for 60 min at 18°C in a TJ-25 centrifuge (Beckman Coulter, Inc., Fullerton, CA) equipped with a TA-15.1.5 rotor. The supernatant was carefully removed and transferred into a PD10 column (Amersham Biosciences, Sunnyvale, CA), in which the lysis buffer was exchanged with Start Buffer (Beckman Coulter, Inc.). Finally, the sample was filtered through sterile low-protein-binding 5.0-μm and 0.45-μm membrane filters (Pall Sciences, Ann Arbor, MI). The protein concentration was determined by MicroBCA assay (Pierce, Rockford, IL) using a DU800 spectrometer (Beckman Coulter, Inc.). Samples A, B, and C had protein concentrations of 0.403, 0.597, and 0.364 mg/mL, respectively. The final volume collected for each lysate was approximately 3.5 mL.

Protein Fractionation

The proteins from all three samples were fractionated independently by pI and hydrophobicity with a ProteomeLab PF 2D Protein Fractionation System (Beckman Coulter, Inc.). The first dimension separated the proteins by chromatofocusing, whereas the second is by reversed-phase chromatography. The first-dimension hardware consists of a manual injector, pump, pH monitor, and UV detector, whereas the second-dimension hardware consists of a binary pump and UV detector. Between the two dimensions, there is a combination fraction collector for the first dimension, which is used as the autosampler-injector for the second dimension. The chemistry components of this system include a chromatofocusing column, a high-performance reversed-phase column, and the start and eluent buffers for the first dimension.

In the first dimension, a chromatofocusing column was set at room temperature with a flow rate of 0.2 mL/min. This module had an injection loop of 2.0 mL. Before injection, the column was equilibrated with start buffer (pH 8.5) for 130 min. The pH electrode was calibrated with pH 4, 7, and 10 buffers before the start of the run. The first-dimension separation was started with the sample injection; in the first 20 min, start buffer was pumped through the column to elute proteins with pI value above 8.5. After 20 min, the pH gradient from 8.5 to 4.0 was started by changing the elution solvent to eluent buffer (pH 4.0). After the pH gradient (115 min), a 1 M sodium chloride (Spectrum, Gardena, CA) solution was used to remove proteins with pI values below 4.0 from the column. The final step was to rinse the column with water. During the first-dimension separation, fractions were collected every 0.3 pH units during the pH gradient and every 7.5 min when the pH value was constant. Data for both pH measurements and absorbance at λ = 280 nm were collected throughout the separation using a data rate of 1 Hz. At the end of the first-dimension separation, the second-dimension separation was started.

The second dimension used a high-performance reversed-phase column with a flow rate of 0.75 mL/min at 50°C. A gradient was formed using 0.1% trifluoroacetic acid (TFA; J.T. Baker, Phillipsburg, NJ) in water and 0.08% TFA in acetonitrile (Burdick & Jackson, Muskegon, MI). The proteins were detected by absorbance at λ = 214 nm (data collection set at 5 Hz), and fractions were collected at intervals of 15 sec using a FC204 fraction collector (Gilson, Middleton, WI). The first-dimension fractions were analyzed by injecting 200 μL of each into the second-dimension column. After all the first-dimension fractions had been analyzed, a proteome map was generated using the ProteoVue software program (Beckman Coulter, Inc.). This program aligns the second-dimension chromatograms with the starting and ending pH values of each collected fraction, generating an image analogous to a 2D gel.

Protein Identification

A second-dimension protein fraction with pI value above 8.5 was identified by trypsin digestion followed by MS analysis. Digestion was carried out as follows: The fraction of interest was evaporated to a volume of 10 μL or less using a Speedvac SC110 (Savant, Holbrook, NY). Its final volume was adjusted to 10 μL with water. Then it was reduced with dithiothreitol by heating at 60°C for 1 h. Next it was alkylated with iodoacetamide and incubated at 25°C for 30 min. Finally, the protein fraction was digested with trypsin and incubated at 37°C for 24 h.

MALDI-TOF MS analysis was done with a Micromass TOFSPEC-2E (Micromass/Waters, Milford, MA) instrument using α-cyanohydroxycinnamic acid as matrix. Positive polarity was used in reflectron mode. The database used in the identification of the trypsin-digested peptides was SwissProt.10.30.2003.

RESULTS AND DISCUSSION

The most desired feature in proteome profiling and fractionation is reproducibility, in which sample preparation plays a critical role. Thus, the first step on this study was to develop a protocol for preparing lysates of bovine skeletal muscle in a consistent way. The procedure given in this paper was tested for consistency by having different individuals perform it on different days using aliquots from the same muscle sample. The protocol was deemed reliable because all three samples gave 1–2 mg of protein for 100-mg muscle samples. In addition, all three lysates gave reproducible proteome profiles, as discussed below.

Figure 1​1 shows the proteome profiles generated by each bovine skeletal muscle lysate. The pI values increase from left to right, whereas hydrophobicity increases from bottom to top. Thus, protein location within the proteome profile is directly linked to its physical properties. The first 6 min of each profile are not shown because high amounts of UV-absorbing salt from the first-dimension separation eluted during the first 2 min and there were no significant peaks in the next 4 min. No IEF markers were needed because the eluent pH was measured throughout the first-dimension separation. Based on the results with protein standards, the standard deviation in the pH measurement is ±0.1 pH units, while the standard deviation of the second-dimension retention time is ±0.2 min (results not shown). The horizontal line across each proteome profile with a retention time of 13.5 min was produced by a UV-absorbing impurity present in the acetonitrile used in the second-dimension separation. In general, all three profiles are highly complex and they proved to be independent of sample amount and instrumentation.

Proteome profiles of bovine skeletal muscle generated by the 2D liquid chromatography system with injections of 725 μg (A), 1194 μg (B), and 728 μg (C) of protein from three separately prepared samples. The numbers at the top of...

The proteome profiles of samples A and B (Figures 1A and B​B,, respectively) were generated by the same 2D liquid chromatography system, but they differ in the amount of protein injected. A total of 725 μg of sample A proteins were injected, whereas 1194 μg of sample B were injected. Both profiles are similar, but more protein peaks were observed for sample B (1046 peaks) than for sample A (954 peaks), which follows from the difference of injected protein mass. It is important to mention that the maximum loading capacity of this 2D liquid chromatographic system (5 mg) has not been reached with 1.2 mg of protein.

The proteome profile of sample C (Figure 1C​1C)) was obtained by injecting 728 μg of protein, which is practically the same amount of protein used to generate the sample A profile. However, the proteome profile of sample C was generated using a different protein fractionation system, columns, and start and eluent buffers than those used in profiling samples A and B. The main difference in all three proteome profiles is that the protein peaks in sample C are shifted by at most one lane when compared with those of samples A and B. This shift was caused by a difference in the start of the pH gradient between samples A and B and sample C. In the method used for all three profiles, the fraction collector was programmed to start collecting by intervals of 0.3 pH units when the pH value at the start of the fraction is 8.4 or below. Because of this, the fractionation by pH for samples A and B was started in lane 18, whereas sample C was started in lane 19. Another profile discrepancy is that the pH gradient ended at different lanes: lane 5 for samples A and B, and lane 6 for sample C. Nevertheless, the pI and retention time values of sample C proteins are consistent with those obtained for samples A and B. A total of 927 peaks were detected in the sample C profile, which is comparable to the 954 peaks observed for sample A. Overall, the three proteome profiles are consistent.

The fact that protein fractions are collected in liquid form is another advantage of using a 2D protein fractionation system for proteome profiling. Sample handling is minimized, thus reducing the risk of sample loss and degradation. However, the proteome profiles are complex, and identifying over 900 fractionated proteins is difficult.

In this study, MALDI-TOF-MS was used to identify one of the proteins by its tryptic digestion peptide fragments and to test our procedures. The protein from lane 24 with retention time of 17–18 min was chosen because it is present in large quantity and its pI value above 8.5 ensures that it has never been reported in 2D gels. Figure 2​2 shows the MALDI spectrum obtained for this protein fraction. The top 10 prospects from the database search identified it as lysozyme C, with a maximum of 9 out of 17 peptides matched.

MALDI-TOF MS analysis obtained from the tryptic digest of lane 24, with retention time of 17–18 min. This protein was identified as lysozyme C.

Lysozyme is an enzyme with antibiotic properties, a pI value of 10.5–11.0, known to be present in a great number of tissues and secretions.4 The work of Moss et al.5 describes the extraction and purification of four lysozyme isoforms from bovine cartilage using three chromatographic techniques. However, the normal amount of lysozyme present in bovine skeletal muscle is unknown. According to the proteome profiles generated in this work, lysozyme is fairly abundant in bovine muscle. Work is in progress in our laboratory to develop methods that include quantitation as well as identification of all fractionated proteins of bovine skeletal muscle.

Acknowledgments

We would like to thank Mr. Chul Yoo and Dr. David M. Lubman from the University of Michigan at Ann Arbor for the MALDI-MS analysis.